1. A two-state homology model of the hERG K + channel: application to ligand binding Ramkumar Rajamani, Brett Tongue, Jian Li, Charles H. Reynolds J & J PRD Biorg. Med. Chem. Lett. 2005, 15, 1737 Kiran-20060214

2. There is a wide range of anticancer, antipsychotic, antihypertensive and antihistamine compounds which interact with the hERG channel. hERG plays a critical role in repolarization of the cardiac action potential and interference leads to serious side effects. The hERG K + channel is a tetramer. Each monomer has 6 transmembrane domains. Voltage dependent opening and closing is determined by the S4 domain. The S6 domain has a cavity where drugs bind. Biology

3. Defining the problem No crystal structure available. The closest homologous protein is the bacterial KcsA protein which has been crystallized. We are dealing with not one, but four protein molecules which co-assemble to form a functional channel. The channel has open and closed states, the issue of flexibility has to be addressed. Once a ligand binds, the S6 helix can close to varying degrees. - Acronyms: Two crystal structures were used for homology modeling: KcsA from Streptomyces lividans and MthK from Methanobacterium thermoautrophicum

4. Goal and Methods Goal: Identify specific protein-ligand interactions responsible for affinity to hERG and predict ligand modifications to mitigate binding Method: - Align sequences in the pore forming region, then predict coordinates for non-identical residues using PRIME. Add hydrogens and charges using Maestro, do a constrained minimization of the protein. - Dock the ligand set using GLIDE. The best pose for each ligand is minimized within the protein using a conjugate gradient minimizer (0.05), the OPLS-AA force field and the GB/SA continuum water model, residues within 8 Å are also allowed to optimize. - The ligand from the minimized ligand-receptor complex is extracted and re-minimized in GB/SA water to get reference energetics for the free state. The LIE (Linear Interaction Energy) method was used to predict binding affinities. - It was determined that multiple homology models were needed to mimic the partially closed and open states of the channel. The KcsA and the MthK structures were used.

5. Results Initial homology modeling with the (closed) KcsA structure revealed that the S6 domain active site cavity was too small to accommodate known ligands of hERG. The hERG K + structure contained a Phe656 residue in an equivalent position to the Thr residue in KcsA. The difference in the size of the two residues argued for a channel structure that differed in the S6 helix domain from that of the closed KcsA structure. This led to an examination of another known crystal structure: the MthK structure (in open state) was known.

6. - Glycine in the hinge is conserved across species, the direction and angle of the bend was used to derive intermediate states of helix motion Closed channel Open channel Copyright © 1999-2005 Joyce J. Diwan. at RPI

7. The S6 helix from the reference closed state was rotated to match the reference open state. The channel was closed in 1° increments and subjected to a protocol of heating (0.4 ps), equilibration (0.6 ps), dynamics (5 ps). A harmonic constraint of 24 kcal/mol/Å 2 was applied on the C  atoms. This protocol removed any unfavorable interactions arising from helix translation. Derivation of two states for docking studies

8. Results In the dual state model, ligands were separated into bins based on their preference for a particular state (open 19° tilt, partially open 10° tilt) The preference was established by comparing (∆ele + ∆vdw) for each ligand in the two states and picking the lower value. A set of 32 ligands (for which experimental IC50s were known) was examined. All 32, R 2 = 0.24 21, preference for open state, RMSD = 1.2 11, preference for partially open, RMSD = 0.85

9. Equations: Combine energies for each ligand from its best fit state, 5 outliers found Primary contribution is from the vdw term Results 27 compounds, R 2 = 0.82 RMSD = 0.56 pIC 50open = -0.166(∆vdw) + 0.002(∆ele) (1) pIC 50partiallyopen = -0.155(∆vdw) + 0.004(∆ele) (2) pIC 50combined = -0.163(∆vdw) + 0.0009(∆ele) (3)

10. The combined two-state model of hERG binding affinity could reproduce pIC50 values and correct docked poses. This study improved upon earlier work with single state models. The hydrophobicity of Phe656 and Tyr652 was the best predictor of affinity. Conclusions